BAX is a member of the BCL-2 family that can promote cell death by making possible the release of apoptotic factors from mitochondria and the endoplasmic reticulum. Many different proteins have been identified that regulate the activity of BAX, but the conformational changes that enable the translocation of BAX to membranes remain unknown. Hence a fundamental gap in the knowledge base exists - there is no unifying mechanism that reconciles how the interaction of BAX with many distinct regulatory factors controls its ability to bind to membranes. To address this, the long-term goals of the proposed studies are to elucidate the means by which BAX interacts with membranes and understand how this interaction modulates the protein's function. The objective of this application is to determine how occupancy of a prominent hydrophobic groove regulates the capacity of BAX to associate with membranes. The central hypothesis is that the C-terminal 19 helix of BAX can bind in two different orientations, forward or reverse, within the hydrophobic groove, and that the orientation of binding regulates stability of the groove and thereby the ability of BAX to bind to membranes, a process that is enhanced by a transient apoptotic alkalinization mediated by the sodium hydrogen exchanger (NHE). The rationale for the proposed research is that understanding how occupancy of the hydrophobic groove by the 19 helix mediates the membrane translocation of BAX can potentially lead to the development of an effective approach for the pharmacological manipulation of the protein in disease states. The proposed research is relevant to that part of NIH's mission that involves developing fundamental knowledge with the goal of reducing the burden of human disease. Supported by strong preliminary data, the central hypothesis will be tested by pursuing the following three specific aims: (1) Identify the molecular interactions that regulate the binding of the C-terminal 19 helix of BAX within the hydrophobic groove;(2) Determine how occupancy of the hydrophobic groove modulates the membrane association of BAX;and (3) Establish how intracellular alkalinization enhances the binding of BAX to membranes. To achieve the first aim, mutagenesis of a critical site in the hydrophobic groove will be used to examine molecular interactions with the 19 helix. To achieve the second aim, dimerization of BAX will be assessed in conjunction with the exposure of novel transmembrane domains. To achieve the third aim, lipid diffusion in membranes will be examined in the context of NHE defective cells. The research design is innovative because it involves an interdisciplinary approach, uniting biophysical measurements with functional assays to determine how BAX translocates to membranes. The proposed research is significant because, by demonstrating that the stability of the hydrophobic groove controls the accessibility of transmembrane domains, we will have revealed a previously unknown function for the groove. This finding will advance work in the field, leading to the development of novel strategies for the therapeutic manipulation of BAX in human diseases for which dysregulation of this protein figures prominently. The proposed research will fill an existing gap in the knowledge base by revealing the mechanism underlying key conformational changes that enable BAX to transition to membranes. The health benefits derived from the proposed studies will include the potential to therapeutically manipulate the activity of BAX in disease states: turning off the apoptotic activity of BAX to prevent the death of neurons that cause neurodegeneration or cardiomyocytes that cause heart damage or turning on the apoptotic activity of BAX to sensitize cancerous cells to chemotherapy. Results from the proposed studies will improve the fundamental understanding of how proteins of the BCL-2 family interact with membranes, a general finding with broad application in the improvement of the health of human beings.